Localized cutaneous leishmaniasis (LCL) in India is due mostly to Leishmania tropica. It is mainly endemic in the deserts of Rajasthan. Recently, Himachal Pradesh has been identified as a new endemic focus for the disease. In the last few years, the number of new cases has been increasing almost to epidemic proportions. This report presents the preliminary findings of clinico-epidemiologic and investigative results of 161 new localized cases of LCL seen between May 2001 and December 2003. The study populaton was composed of 80 males and 81 females between 10 months and 75 years of age. All were indigenous to the sub-alpine valley along the Satluj River in the mountainous region of the Kinnaur District (altitude = 700-2,900 meters). Most patients were seen from April to September and had 1-8 lesions (duration = 1-6 months) that involved mainly the face. Tissue smears were positive for amastigotes in 37% and histopathology showed non-caseating epitheloid cell granuloma in 77% of the cases. Analysis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the ribosomal gene region of 10 biopsy specimens showed amplicons indistinguishable from L. donovani in eight cases and L. tropica in two cases. Leishmania was cultured on modified Nicole-Novy-McNeal (NNN) medium containing RPMI 1640 medium and heat-inactivated fetal bovine serum from 13 of 38 biopsy samples. Three of these isolated strains were identified as L. donovani while a fourth was L. tropica by PCR-RFLP of the ribosomal internal transcribed spacer region. One strain had a gp63 sequence identical to that of east African strains. Another strain had a unique gp63 sequence that has not been found in L. donovani complex strains. Sand flies trapped in the cattle sheds of a few patients were identified as Phlebotomus longiductus (Parrot 1928). Treatment with intralesional sodium stibogluconate was effective in all patients without any major side effects. One patient developed lupoid leishmaniasis that responded to higher dose of sodium stibogluconate. Though rarely reported as a cause of LCL, L. donovani seems to be the predominant pathogen in this new focus of cutaneous leishmaniasis. Phlebotomus longiductus is a possible vector, albeit based on circumstantial evidence.

Treatment of multidrug-resistant tuberculosis has become one of the major problems in public health. Understanding the molecular mechanisms of drug resistance has been central to tuberculosis research in recent times. DNA microarray technology provides the platform to study the genomic variations related to these mechanisms on a comprehensive level. To investigate the role of efflux pumps in drug resistance, we have constructed a custom DNA microarray containing 25 drug efflux pump genes of Mycobacterium tuberculosis (Indian Patent file no. 2071/DEL/2007) and monitored changes in the expression of these genes on exposure of common anti-tuberculous drugs. Expression profiling of efflux pump genes in multidrug-resistant M. tuberculosis isolates showed overexpression of 10 genes following exposure to various anti-tuberculous drugs. Although two of these genes (Rv3065 and Rv2938) have already been reported to be active drug efflux pumps in M. tuberculosis in earlier studies, the increased activities of other eight efflux pump genes (Rv1819, Rv2209, Rv2459, Rv2477c, Rv2688, Rv2846, Rv2994, and Rv3728) have been demonstrated in multidrug-resistant isolates by us for the first time. After confirmation of differential expressions of these genes by real-time reverse transcription polymerase chain reaction, it was observed that a simultaneous overexpression of efflux pump genes Rv2459, Rv3728, and Rv3065 was associated with resistance to the combination of isoniazid and ethambutol, and these drugs, along with streptomycin, were identified to group together, where efflux-mediated drug resistance appears to be important in M. tuberculosis and follows a constant pattern of induction in multidrug-resistant isolates. Isoniazid and ethambutol combination was also found to be affected in 10% (6/60) of the clinical isolates in the presence of carbonyl cyanide m-chloro phenylhydrazone in resazurin microtitre plate assay, supporting the role of efflux pumps in the resistance to these drugs. Overexpression of two of the genes (Rv2477 and Rv2209) has also been observed with ofloxacin stress in M. tuberculosis.

Molecular epidemiologic findings suggest an ancient focus of TB.

Mycobacterium bovis, the causative agent of bovine tuberculosis, has no known geographical boundaries. M. bovis infection occurs in diverse groups of animals, which include farm animals of economic importance, wildlife, and humans (1,16,17,31). Despite these ominous features of M. bovis, to date there have been only projected global estimates of the disease burden. There has been no international effort to determine the actual disease burden, owing to the nonavailability of a reliable user-friendly technology for early detection of M. bovis in clinical samples. Bovine tuberculosis has been on the increase in developed countries and continues to occur in developing countries (6, 14, 16). In Africa, approximately 85% of cattle and 82% of the human population live in areas where the disease is prevalent (6, 26). There are limited reports from India (24, 27, 38, 40) and from underdeveloped countries (6, 16) relating to the prevalence of and infection with M. bovis in cattle. Detection of M. bovis in bovine samples has become necessary, as infected animals are potentially capable of infecting humans (zoonotic tuberculosis) (6). Besides M. bovis, transmission of M. tuberculosis from infected humans to animals and back has been reported (reverse zoonosis) (10,12,41). Hence, M. bovis and M. tuberculosis pose a potential health hazard to both animals and humans (1,15,43).The identification of the closely related members of the Mycobacterium tuberculosis complex (MTC) has remained a challenging task in diagnostic laboratories (7,30,44). MTC includes a variety of closely related mycobacteria, namely, M. tuberculosis, M. bovis, M. canetti, M. africanum, and M. microti. A panel of classical tests based on microbiological features such as growth rate and phenotypic and biochemical characteristics has conventionally been utilized to distinguish members of MTC (30). However, these tests are slow, cumbersome, unreliable, and time-consuming. The high degree of variability among these tests warrants the development of molecular biological tools for identification of MTC members. In this regard, multiple gene targets have been used to date to detect and differentiate genetically identical species, such as M. tuberculosis and M. bovis. The gene targets include pncA (2,29,36), gyrB (5), oxyR (42), and katG (19). Huard et al. (21) and Richter et al. (33) have targeted multiple loci and genes to differentiate M. tuberculosis from M. bovis. However, to date no single accepted protocol(s) that can unambiguously differentiate all members of the MTC is available. Identification of the etiological agent belonging to MTC is important for determination of the origin and reservoirs of infection and also for implementation of appropriate public health measures.Our laboratory earlier described a PCR-restriction fragment length polymorphism (RFLP) method utilizing the hupB gene,

Prolonged treatment of tuberculosis (TB) often leads to poor compliance, default and relapse, converting primary TB patients into category II TB (Cat IITB) cases, many of whom may convert to multi-drug resistant TB (MDR-TB). We have evaluated the immunotherapeutic potential of Mycobacterium indicus pranii (MIP) as an adjunct to Anti-Tubercular Treatment (ATT) in Cat II pulmonary TB (PTB) patients in a prospective, randomized, double blind, placebo controlled, multicentric clinical trial. 890 sputum smear positive Cat II PTB patients were randomized to receive either six intra-dermal injections (2 + 4) of heat-killed MIP at a dose of 5 × 108 bacilli or placebo once in 2 weeks for 2 months. Sputum smear and culture examinations were performed at different time points. MIP was safe with no adverse effects. While sputum smear conversion did not show any statistically significant difference, significantly higher number of patients (67.1%) in the MIP group achieved sputum culture conversion at fourth week compared to the placebo (57%) group (p = 0.0002), suggesting a role of MIP in clearance of the bacilli. Since live bacteria are the major contributors for sustained incidence of TB, the potential of MIP in clearance of the bacilli has far reaching implications in controlling the spread of the disease.

Pyrazinamide (PZA) is an important first-line antituberculosis drug because of its sterilizing activity against semidormant tubercle bacilli. In spite of its very high in vivo activity, its in vitro activity is not apparent unless an acidic environment is available, which makes PZA susceptibility testing difficult by conventional methods. The present study was, therefore, planned to assess the performance of the colorimetric BacT/ALERT 3D system and compare the results with those from conventional tests, i.e., the Löwenstein-Jensen (LJ) proportion method (pH 4.85) and Wayne's pyrazinamidase (PZase) assay, using 107 clinical isolates. The concordance among all of these tests was 89.71% after the first round of testing and reached 92.52% after resolution of the discordant results by retesting. Prolonged incubation of the PZase tube for up to 10 days was found to increase the specificity of the PZase test. The concordances between LJ proportion and BacT/ALERT 3D, LJ proportion and the PZase assay, and BacT/ALERT 3D and the PZase assay were found to be 99.06%, 93.46%, and 92.52%, respectively. Using the LJ results as the gold standard, the sensitivities of BacT/ALERT 3D and the PZase assay were 100 and 82.85%, respectively, while the specificity was 98.61% for both of the tests. The difference between the sensitivities of BacT/ALERT 3D and the PZase assay was significant (P ‫؍‬ 0.025). The mean turnaround times for the detection of resistant and susceptible results by BacT/ALERT 3D were 8.04 and 11.32 days, respectively. While the major limitations associated with the PZase assay and the LJ proportion method are lower sensitivity in previously treated patients and a longer time requirement, respectively, the BacT/ ALERT 3D system was found to be rapid, highly sensitive, and specific.

Whole genome sequencing (WGS) of Mycobacterium tuberculosis has been constructive in understanding its evolution, genetic diversity and the mechanisms involved in drug resistance. A large number of sequencing efforts from across the globe have revealed genetic diversity among clinical isolates and the genetic determinants for their resistance to anti-tubercular drugs. Considering the high TB burden in India, the availability of WGS studies is limited. Here we present, WGS results of 200 clinical isolates of M. tuberculosis from North India which are categorized as sensitive to first-line drugs, mono-resistant, multi-drug resistant and pre-extensively drug resistant isolates. WGS revealed that 20% of the isolates were co-infected with M. tuberculosis and non-tuberculous mycobacteria species. We identified 12,802 novel genetic variations in M. tuberculosis isolates including 343 novel SNVs in 38 genes which are known to be associated with drug resistance and are not currently used in the diagnostic kits for detection of drug resistant TB. We also identified M. tuberculosis lineage 3 to be predominant in the northern region of India. Additionally, several novel SNVs, which may potentially confer drug resistance were found to be enriched in the drug resistant isolates sampled. This study highlights the significance of employing WGS in diagnosis and for monitoring further development of MDR-TB strains.

Mycobacterium tuberculosis, the causative agent of tuberculosis, accounts for 1.5 million human deaths annually worldwide. Despite efforts to eradicate tuberculosis, it still remains a deadly disease. The two best characterized strains of M. tuberculosis, virulent H37Rv and avirulent H37Ra, provide a unique platform to investigate biochemical and signaling pathways associated with pathogenicity. To delineate the biomolecular dynamics that may account for pathogenicity and attenuation of virulence in M. tuberculosis, we compared the proteome and phosphoproteome profiles of H37Rv and H37Ra strains. Quantitative phosphoproteomic analysis was performed using high-resolution Fourier transform mass spectrometry. Analysis of exponential and stationary phases of these strains resulted in identification and quantitation of 2709 proteins along with 512 phosphorylation sites derived from 257 proteins. In addition to confirming the presence of previously described M. tuberculosis phosphorylated proteins, we identified 265 novel phosphorylation sites. Quantitative proteomic analysis revealed more than five-fold upregulation of proteins belonging to virulence associated type VII bacterial secretion system in H37Rv when compared to those in H37Ra. We also identified 84 proteins, which exhibited changes in phosphorylation levels between the virulent and avirulent strains. Bioinformatics analysis of the proteins altered in their level of expression or phosphorylation revealed enrichment of pathways involved in fatty acid biosynthesis and two-component regulatory system. Our data provides a resource for further exploration of functional differences at molecular level between H37Rv and H37Ra, which will ultimately explain the molecular underpinnings that determine virulence in tuberculosis.